Immobilized Acylase PvdQ Reduces Pseudomonas aeruginosa Biofilm Formation on PDMS Silicone

Jan Vogel,Rita Setroikromo,Marijke Wakker-Havinga,Wim J Quax

doi:10.3389/fchem.2020.00054

Abstract

The bacterial biofilm plays a key role in nosocomial infections, especially those related to medical devices in sustained contact with patients. The active dispersion of bacterial cells out of biofilms acts as a reservoir for infectious diseases. The formation of such biofilms is a highly complex process, which is coordinated by many regulatory mechanisms of the pathogen including quorum sensing (QS). Many bacteria coordinate the expression of key virulence factors dependent on their population density through QS. The inhibition of this system is called quorum quenching (QQ). Thus, preventing the development of biofilms is considered a promising approach to prevent the development of hard to treat infections. Enzymatic QQ is the concept of interfering with the QS system of bacteria outside the cell. PvdQ is an acylase with an N-terminal nucleophile (Ntn-hydrolase) that is a part of the pyoverdine gene cluster (pvd). It is able to cleave irreversibly the amide bond of long chain N-acyl homoserine lactones (AHL) rendering them inactive. Long chain AHLs are the main signaling molecule in the QS system of the gram-negative pathogen Pseudomonas aeruginosa PA01, which is known for surface-associated biofilms on indwelling catheters and is also the cause of catheter-associated urinary tract infections. Furthermore, PA01 is a well characterized pathogen with respect to QS as well as QQ. In this study, we immobilized the acylase PvdQ on polydimethylsiloxane silicone (PDMS), creating a surface with quorum quenching properties. The goal is to control infections by minimizing the colonization of indwelling medical devices such as urinary catheters or intravascular catheters. The enzyme activity was confirmed by testing the degradation of the main auto-inducer that mediates QS in P. aeruginosa. In this article we report for the first time a successful immobilization of the quorum quenching acylase PvdQ on PDMS silicone. We could show that immobilized PvdQ retained its activity after the coating procedure and showed a 6-fold reduction of the auto-inducer 3-oxo-C12 in a biosensor setup. Further we report significant reduction of a P. aeruginosa PA01 biofilm on a coated PDMS surface compared to the same untreated material.

Highlights

The bacterial colonization of surfaces is an omnipresent observation in almost every environment
As a proof of principle to confirm the activation of the polydimethylsiloxane silicone (PDMS) surface as well as the attachment of the acylase PvdQ to this activated surface we performed the following experiment before proceeding to activity assays
Studies have shown that even though the formation of a bacterial biofilm is not solely depending on quorum sensing (QS) but involves a multitude of different regulatory networks, QS systems play an important role in the formation and differentiation of a mature P. aeruginosa biofilm (Stoodley et al, 2002)

Summary

Introduction

The bacterial colonization of surfaces is an omnipresent observation in almost every environment. In healthcare settings biofilms formed by pathogenic bacteria represent a constant threat to the patient’s health (Fux et al, 2005). Hospitals follow strict hygiene protocols to keep the clinical environment free of bacteria, reports show that pathogens can persist for months on surfaces (Kramer et al, 2006). Surgical equipment and surfaces need to be sanitized and sterilized, as for example can be seen from the extremely persistent pathogen Acinetobacter baumannii. It could be shown that surface attached Acinetobacter could withstand dehydration and surface sanitization by ethanol (Harding et al, 2017). Together with Pseudomonas aeruginosa this pathogen is a cause for ventilator associated infections (Sievert et al, 2013)

Methods

Results

Conclusion